Does Malfunction of Arachidonic Acid Epoxygenase Explain Salt-Sensitive Hypertension?
Authors: K. Nakagawa, V.R. Holla, Y. Wei, W.H. Wang, A. Gatica, S. Wei, S. Mei, C.M. Miller, D.R. Cha, E. Price, R. Zent, A. Pozzi, M.D. Breyer, Y. Guan, J.R. Falck, M.R. Waterman, J.H. Capdevila
Journal: Journal of the American Society of Nephrology (2006)

Historians claim that the relation between high salt intake and high BP was known even to the ancient Chinese, as exemplified by the writings of the Yellow Emperor (1): “hence if too much salt is used in food, the pulse hardens, tears make their appearance and the complexion changes ”. But it was the experiment of controlled sodium chloride loading by Ambard and Beaujard (2) that provided the first evidence of a link between salt loading and BP. The groundbreaking work of A. Guyton led to the modern concept that a disturbed BP/natriuresis relationship underlies any form of hypertension (3,4), i.e., that kidney malfunction—not necessarily primary kidney disease—is the reason why higher BP values are required to get rid of the dietary intake of sodium. Clinical studies documented that at least a proportion of patients with primary hypertension are “salt sensitive” (5), but even today there is no consensus on the method to identify “salt sensitivity.” The finding that practically all monogenetic forms of hypertension are associated with increased renal tubular sodium reabsorption (6–8) immediately made tubular sodium transporters hot candidates for the genetic predisposition of the garden variety of primary hypertension, which is presumably polygenetic; so far the yield of these studies had been underwhelming. Although there is also some evidence for abnormalities of proximal tubular handling of sodium in primary hypertension (9), one particularly attractive candidate was ENaC (epithelial sodium channel), the epithelial cell sodium transporter in the most distal part of the nephron, the collecting duct, where the fine tuning of urinary sodium excretion is thought to take place. But primary hypertension stubbornly refused all efforts to link it to mutations of the ENaC analogous to the mutation of which had been found in the rare Liddle syndrome (10). Nevertheless, the concept to link primary hypertension, or at least the salt-sensitive varieties of primary hypertension, to ENaC malfunction appeared a priori plausible. Apart from direct mutations in the ENaC molecule, mutations coding for molecules modulating ENaC activity remained a possibility and it is here where the above authors struck gold. The authors had previously shown that mice with disruption of the CYP4a14 gene develop androgen-sensitive hypertension and formation of the prohypertensive 20-hydroxyarachidonate (11). After previous suggestions by McGiff (12), Nakagawa et al. had concluded that Cyp4a arachidonate monooxygenase plays a role in the renal regulation of BP. At this point it is necessary to digress for a moment for some comments on arachidonic acid metabolism. Arachidonic acid is metabolized by a group of cytochrome P450 (CYP; http://www.cypalleles.ki.se/) epoxygenases and ω-hydroxylases, which generate epoxyeicosatrienoic acids (EET) and 20-hydroxyeicosatetraenoic acid (20-HETE). In this study Nakagawa et al. had produced mice with disruption of cytochrome P450 family 4, subfamily a, polypeptide 10 (Cyp4a10−/−) (13). These mice developed normally and showed no evidence of organ malformation or disease. The phenotype was characterized by salt-sensitive hypertension, high salt intake causing sodium retention and fluid expansion (by nuclear magnetic resonance measurements); renal morphology and gross renal function were normal before and after salt loading. The knockout animals were normotensive on 0.05% salt (low salt), though in the knockout (both male and female), but not in the wild-type animals, BP increased substantially when they were switched to 0.3% (normal salt) and 8% (high salt), respectively. How did the disruption of Cyp4a10 affect synthesis of 20-HETE and EET? In a nutshell, using appropriate experiments the authors (13) could exclude 20-HETE as the culprit, in line with previous studies showing that it is only another CYP enzyme, i.e., CYP4a12, which has significant 20-HETE synthase activity (14). In contrast, EET appeared to be of interest, since it was known to be an antihypertensive molecule (15,16), the synthesis of which is sensitive to dietary salt intake (17). Inhibition of its synthesis was known to cause salt-sensitive hypertension (18). The logical next step was to test not only whether in the knockout mice BP was salt-sensitive, but also to test whether it was responsive to amiloride. Reversal of hypertension by amiloride, which acts on the collecting duct sodium channels, would indicate that ENaC dysfunction accounts for the hypertensive phenotype of the CYP4a10−/−—amiloride reversed hypertension on high salt; thus, the smoking gun pointed to ENaC! Indeed, recent evidence pointed to a role of EET in the control of ENaC function. In a patch-clamp study of the rat cortical collecting duct, Wei et al. (19) had recently shown that the precursor substance arachidonic acid decreased the activity of ENaC. This effect was specific and not mimicked by nonmetabolizable analogs of arachidonic acid. It was also not abrogated by inhibition of cyclooxygenase or of CYP450ω hydroxylation, while a specific inhibitor of the CYP epoxygenase activity clearly nullified the effect of arachidonic acid on ENaC. The downstream product 11,12-EET, but not EET isomers, reduced ENaC activity. From these findings Wei et al. concluded that the CYP-epoxygenase product 11,12-EET was the culprit. Nakagawa et al. (13) carried this one step further by comparing the effects of arachidonic acid and 11,12-EET on ENaC activity in the cortical collecting ducts of the CYP4a10 wild-type and knockout mice. In the CYP4a10−/− knockout mice, basal ENaC activity was increased and, in contrast to the wild-type mice, such increased activity was virtually refractory to inhibition by arachidonic acid. In contrast to arachidonic acid in both wild-type and knockout mice, 11,12-EET was able to inhibit ENaC in the cortical collecting duct. This finding identifies lack of production of 11,12-EET by the CYP arachidonic acid monooxygenase as the crucial hypertensinogenic pathomechanism. This hypothesis is directly supported by measurements of the metabolite 11,12-EET in the urine. Phenomenologically the ENaC behaved as if the ENaC molecule had a gain-of-function mutation (which is the cause of the Liddle syndrome): The channel activity (open probability × channel number) was constitutively increased in the CYP4a10−/− mice, but was reduced when the assumed metabolic block was sidestepped by directly presenting the suspected inhibitory metabolite 11,12-EET. A final twist was an observation implying interaction between peroxisome proliferator-activated receptor α (PPARα) and EET. Why should PPARα be of interest in this context? It had been known that PPARα activation can lower BP (15,20), particularly salt-dependent hypertension during chronic endothelin B receptor blockade (21), and that PPARα causes changes in the renal expression of the CYP4A isoforms (15). A selective PPARα agonist, Wyeth 14643, was administered to CYP4a10+/+ wild-type and CYP4a10−/− mice to measure 11,12 epoxygenase metabolites in the urine. The long and the short of it is that Wyeth 14643 increased renal biosynthesis of EET in both CYP4a10+/+ wild-type and CYP4a10−/− mice. It also normalized BP in hypertensive animals. This finding is of interest, since fibrates (such as PPARα agonists) have so far been viewed only as lipid-lowering agents. Antihypertensive effects had not generated much interest and deserve further study. Another conclusion from the findings would also be to view EET and epoxygenases as attractive candidates for potential antihypertensive interventions—if the animal data can be extrapolated to humans. Indeed the observation of Laffer et al. (22) pointed to a role of another arachidonic acid product, HETE, in modulating salt sensitivity by natriuretic mechanisms. Furthermore, an association was documented between hypertension and the T8590C polymorphism of the Cyp4a11 gene in the Augsburg substudy of the MONICA project (23), in line with the data from the United States by Gainer et al. (24). It is likely that this monooxygenase plays a role in salt-sensitive hypertension of humans as well, at least in whites. Primary hypertension is certainly heterogeneous, as illustrated, to give one example, by the different behavior of nephron numbers in hypertensive whites and in blacks, respectively: It is low in white (25,26) but not black (26) hypertensives, and in this context it is also of interest that a polymorphism in another CYP enzyme, CYP3A5, which plays a role in cortisol metabolism, has been shown to be associated with salt sensitivity and hypertension in blacks (27,28) Finally, from a public health perspective, the expected results in this area may be of considerable relevance to provide a rationale for reduction of salt intake in the general population. Despite the clear demonstration of efficacy in the DASH (Dietary Approaches to Stop Hypertension) study (29), implementation meets considerable resistance for nonmedical reasons—to put it politely (30).